Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

Ren, Rui; Gao, Jie; Yin, Dongmei; Li, Kai; Lu, Chuqiao; Ahmad, Sagheer; Wei, Yonglu; Jin, Jianpeng; Zhu, Genfa; Yang, Fengming

doi:10.3389/fpls.2021.626015

Cited by 42 publications

(42 citation statements)

References 81 publications

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“…Protoplast isolation and transfection from the leaf bases of Cymbidium sinense was conducted based on protocols established by Ren et al . ( 2021 ). Empty vectors pSPYNE‐35S (harbouring the nitrogen terminal of the yellow fluorescent protein [YFP]) and pSPYCE‐35S (harbouring the carbon terminal of the YFP) were both driven by the CaMV 35S promoter.…”

Section: Methodsmentioning

confidence: 99%

The genome of Cymbidium sinense revealed the evolution of orchid traits

Yang

Gao

Wei

et al. 2021

Plant Biotechnology Journal

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Summary The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map for Cymbidium sinense, a well‐known species belonging to genus Cymbidium that has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high‐quality chromosome‐scale genome assembly was 3.52 Gb in size, 29 638 protein‐coding genes were predicted, and evidence for whole‐genome duplication shared with other orchids was provided. Marked amplification of cytochrome‐ and photosystem‐related genes was observed, which was consistent with the shade tolerance and dark green leaves of C. sinense. Extensive duplication of MADS‐box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species‐specific flower traits, including wild‐type and mutant‐type floral patterning, seasonal flowering and ecological adaption. CsSEP4 was originally found to positively regulate gynostemium development. The CsSVP genes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low‐temperature‐dependent flowering. Important genetic clues to the colourful leaf traits, purple‐black flowers and volatile trait in C. sinense were also found. The results provide new insights into the molecular mechanisms of important phenotypic traits of Cymbidium and its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of orchids.

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Section: Methodsmentioning

confidence: 99%

The genome of Cymbidium sinense revealed the evolution of orchid traits

Yang

Gao

Wei

et al. 2021

Plant Biotechnology Journal

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“…At 72 h after infiltration, stained with fluorescent membrane marker MM 4-64 [N-(3-Triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridiniumdibromide] (AAT Bioquest, United States) for 8 min, GhEXO7_At was co-localized with MM 4-64 in N. benthamiana epidermal cells under fluorescence microscopy ( Vida and Emr, 1995 ; Chen et al, 2015 ). Arabidopsis mesophyll protoplasts were isolated and transfected using the Arabidopsis Protoplast Preparation and Transformation Kit (Coolaber, Beijing, China) as described previously ( Yoo et al, 2007 ; Ren et al, 2021 ). After 6–18 h of culture, Arabidopsis mesophyll protoplasts were observed with a fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.)

Liu

Chen

et al. 2021

Front. Plant Sci.

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Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

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“…The plant protoplast system has been used as a versatile and powerful complex for cell-based experiments in many plant species [ 27 , 28 , 29 ]. The highly efficient protoplast transient expression systems have greatly contributed to the development of various fields of botany, including subcellular localization, protein-protein interaction, transport, signal transduction, and metabolic pathways [ 30 , 31 , 32 , 33 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts

Son

Kwon

2021

Plants

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Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.

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Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

Cited by 42 publications

References 81 publications

The genome of Cymbidium sinense revealed the evolution of orchid traits

The genome of Cymbidium sinense revealed the evolution of orchid traits

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.)

A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts

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